PROJECT SUMMARY/ABSTRACT Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), continues to be one of the world?s deadliest infectious diseases because of several factors, including a) poor adherence to the long-term multidrug therapy, b) latent (or persistent) infection, c) co-infection with HIV and d) drug-resistant Mtb strains that are unaffected by current TB therapies. Similarly, Staphylococcus aureus was recently listed by the WHO as a ?high priority? pathogen in regards to new antibiotic development, due to its increasing prevalence as a major infectious agent both in hospitals and in the community, and to increased virulence and rising resistance against current antistaphylococcal agents. The biosynthesis of coenzyme A (CoA), an essential cofactor relevant to several metabolic processes, contains an emerging set of targets for antibacterial drug design. Small molecule inhibitors of this pathway have shown activity in a cell-based model of latent TB, increasing interest in this set of targets as a potential means attacking the latent pathogen. Similarly, CoA biosynthesis in S. aureus has recently been associated with maintaining the organism under anaerobic conditions, a model for persistent infections. The broad, long-term objective of this project is to develop inhibitors of CoA biosynthesis, which act on the PPCS enzyme activity of Mtb and S. aureus, that show whole cell activity under a range of conditions and potential for further development as clinically useful agents that could be used in combination treatments. This goal has strong foundations in results from in vitro genetic studies demonstrating the vulnerability of the target, and in vivo experiments showing that depletion of PPCS activity is bactericidal and prevents a robust Mtb infection in mice. In S. aureus, a natural product that targets PPCS activity shows highly selective whole cell activity. These results suggest that the PPCS activity is a highly promising target that is tractable to inhibition by small molecules. To develop new whole cell active compounds, we are proposing inhibitor structures based on a well-established strategy that has successfully been used to prepare inhibitors of mechanistically similar enzymes, giving rise to compounds that also showed excellent whole cell activity against Mtb and S. aureus. In addition, we are proposing complementary metabolic activation and prodrug approaches to address the known challenge of preparing compounds that are able to cross the Mtb cell envelope. We will pursue this objective through three Specific Aims: 1) Design and synthesis of Pan-CMP mimics as PPCS inhibitors; 2) Biochemical evaluation of metabolic activation and PPCS inhibition of Aim 1 compounds; and 3) Evaluation of PPCS inhibitors in models of actively growing and persistent Mtb and S. aureus. These aims are formulated to systematically optimize the structure of the inhibitors and to follow their on-target activity and selectivity through enzymatic and whole cell assays, the latter including tests against wild-type Mtb and S. aureus and mutant Mtb strains (to show target specificity), models of latent/persistent growth for both Mtb and S. aureus, and an ex vivo macrophage model (in the case of Mtb).